1. Field of the Invention
The present invention relates generally to fluidic processing and sensor technology. More particularly, it relates to a variety of apparatuses and methods for sensing particle properties in a microfluidic environment. Even more particularly, it relates to novel techniques for microfluidic, impedance sensing of particles.
2. Description of Related Art
Conventional impedance-based sensing of particles is a well-accepted method for the counting and sizing of particles and cells and finds wide application in clinical and veterinary laboratories for the analysis of blood, cell suspensions, and other samples. In operation, the technique passes a carrier fluid in which the particles to be counted are suspended through a small volume channel that usually consists of a hole in a membrane. As particles pass through the channel, the flow of electrical current between electrodes immersed in the carrier fluid on either side of the membrane is perturbed. Electronics in the circuitry that drives the electrodes detect the electrical perturbations as particles pass through the channel, and information about the size of the particles may be inferred from the characteristics of these signals. The strength of the electrical perturbation due to the presence of a particle in the channel depends approximately on the ratio of the particle volume to the channel volume and on the magnitude of the electrical current passing in the channel. Counting the electrical events that occur when a metered volume of suspending medium is drawn through the channel, and then dividing the count by the volume is done to determine the concentration of particles in a sample.
Various sensors have been developed that use impedance. U.S. Pat. No. 5,683,569, which is hereby incorporated by reference, discloses a method of sensing chemicals using a sensing material and an impedance sensor containing a channel separated from an electrode by a gap. By comparing the surface potential to the electrical impedance, the presence of a chemical species may be determined. Surface potential may be used to detect particles at low concentrations and resistance may be used to detect particles at high concentrations.
WO 9804355, which is hereby incorporated by reference, discloses a method for determining the behavior of particles in a chamber subject to a spectrum of different frequency dielectrophoretic fields. An electrical measurement is used to detect impedance fluctuations.
U.S. Pat. No. 6,149,789, which is hereby incorporated by reference, discloses a process for particle manipulation using feedback from impedance sensors. U.S. Pat. No. 6,169,394, which is hereby incorporated by reference, discloses an impedance sensor for the electrical detection of samples in a micro-analysis system; at least one pair of electrodes is used to detect the conductivity or impedance of a sample in a microchannel. Impedance sensors are also disclosed in U.S. Pat. Nos. 6,084,503, 5,569,591 and 5,580,435, each of which is hereby incorporated by reference.
Although such conventional techniques offer at least some advantage, they nevertheless include a number of significant disadvantages. While AC methods have allowed the impedances of particles to be determined at several frequencies, far greater information about the particle properties is available if more measurements over a wider range of frequencies could be accomplished for each particle. In addition, the need to accurately meter a set volume of fluid through a channel during a determination of concentrations places stringent constraints on fluid control mechanisms. This typically results in the need for a specialized fluidics platform that is often more bulky than the sensor and electronics combined. Another problem is that conventional membranes used in particle sensing are prone to blockage, necessitating accessibility for vigorous flushing. To accommodate rapid and convenient accessibility, particle impedance sensors usually take the form of benchtop instruments that are not readily adaptable to automated sample handling or in-line detection applications. Finally, the sensors of conventional instruments are often expensive and difficult to change.
The present disclosure overcomes such disadvantages through the introduction of, among other things, a membrane and the use of novel electronic detection and signal processing methods. The design and methodology of this disclosure invoke principles not previously applied to particle impedance sensor apparatuses to realize robust, multi-frequency impedance sensor capabilities that may eliminate the need for external fluid metering, permit miniaturization, allow in-line operation with other fluidic systems and instruments, and facilitate rapid and potentially automated replacement of sensor elements.
Applications of the present invention are vast and include, but are not limited to any application in which Coulter counters are used, cell and particle counting, cell and particle subpopulation analysis, cell viability analysis, cell and particle concentration analysis, cell differential analysis, medical applications, veterinary applications, bioengineering, food analysis, soil analysis, in-line particle detection in fluidic circuits and systems, detection of bacterial spores and other biological agents of potential use in warfare and terrorism, discrimination of potentially harmful biological agents from non harmful biological cells such as pollen and from inert particulate materials such as dust, smoke, and non-viable cells, detection of responses of cells such as human blood cell subpopulations to biological and chemical agents, and detection and discrimination of bacterial cells and spores (including anthrax) for medical, agricultural, environmental, and bio-warfare and bio-terrorism detection applications.
In one aspect, the invention is an impedance sensor including a sensor electrode, first and second driver electrodes, and a channel. The first and second driver electrodes are coupled to the sensor electrode and driven in counter phase to produce a net output signal of about zero at the sensor electrode. The channel is defined through the sensor electrode and the first and second driver electrodes.
In other respects, the sensor electrode may include copper. The sensor electrode may include a first and second dielectric membrane sandwiching a detector electrode. The first or second dielectric membrane may include polyimide. The first or second dielectric membrane may be laminated. The first and second driver electrodes may contact the first and second dielectric membranes, respectively. The first and second driver electrodes may be driven at multiple frequencies. The first and second driver electrodes may be driven with an alternating current signal. The cross section of the channel may be rectangular. The sensor may also include a programmable fluid processor coupled to the sensor electrode.
In another aspect, the invention is a flow-through impedance sensor including a channel, a composite membrane sensor assembly, and first and second driver electrodes. The channel is for transporting a carrier medium and particles through the impedance sensor. The composite membrane sensor assembly is coupled to the channel and includes a detector electrode sandwiched between first and second dielectric membranes. The first and second driver electrodes are coupled to the channel and are positioned adjacent opposite sides of the composite membrane sensor assembly. The first and second driver electrodes are driven in counter phase to produce: (a) a net output signal of about zero at the detector electrode when no particle is within the impedance sensor; and (b) a non-zero net output signal at the detector electrode when a particle is within the impedance sensor.
In other respects, the first and second driver electrodes may be in contact with the composite membrane sensor assembly. The first and second driver electrodes may be driven at multiple frequencies. The first and second driver electrodes may be driven with an alternating current signal. The impedance sensor may also include a programmable fluid processor coupled to the sensor electrode.
In another aspect, the invention is a method for determining a characteristic of a packet. A fluid containing a packet is flowed through an impedance sensor that includes first and second driver electrodes driven in counter phase to produce a net output signal of about zero at a sensor electrode. Perturbations of the net output signal arising from changes in impedance associated with the presence of the packet within the impedance sensor are measured. The characteristic of the packet are then determined from the perturbations.
In other respects, the characteristic of the packet may include packet size. The characteristic of the packet may include packet transit time through the impedance sensor. The characteristic of the packet may include packet velocity. The characteristic of the packet may include packet concentration. The characteristic of the packet may include a relative displacement within the impedance sensor. The characteristic of the packet may include packet impedance.
In another aspect, the invention is a method for determining a characteristic of a particle. An impedance sensor is provided including a sensor electrode, first and second driver electrodes coupled to the sensor electrode and driven in counter phase to produce a net output signal of about zero at the sensor electrode, and a channel defined through the sensor electrode and the first and second driver electrodes. A multi-frequency drive signal is applied to the first and second driver electrodes. An impedance signal is received from the sensor electrode. In-phase and out-of-phase components of the impedance signal are determined at the frequencies of the drive signal. Changes in the in-phase and out-of-phase components indicative of a particle event are detected. Portions of the impedance signal are analyzed about the particle event to determine the characteristic of the particle.
In other respects, the drive signal may include a composite of separate waveforms of different frequencies, each frequency being an integer multiple of a fundamental frequency. The drive signal may consist of 8 separate sine waves having frequencies f, 2f, 4f, 8f, 16f, 32f, 64f, and 128f. The impedance signal components may be represented as 24 bit words. The method may also include deriving a composite signal comprising a moving sum of magnitudes of changes of the in-phase and out-of-phase components. Detecting changes indicative of a particle event may include determining when the composite signal exceeds a threshold value above a noise floor. Analyzing portions of the impedance signal about the particle event to determine the characteristic of the particle may include: calculating an overlap integral, constraining curves associated with the in-phase and out-of-phase components to obey a Kramers-Kronig relationship, determining a velocity of the particle, determining a mean fluid velocity, determining a concentration of particles, determining a size of the particle, determining a relative displacement of the particle, determining a dielectric property of the particle, determining a conductivity property of the particle, determining an impedance of the particle, determining a cell membrane permittivity of the particle, and/or determining a cytoplasmic permittivity of the particle.